Method for producing beer

ABSTRACT

The present invention provides a method for producing beer comprising filtering beer through a porous membrane until such time that the porous membrane is in need of cleaning, contacting the porous membrane with an enzyme selected from the group consisting of cellulases, amylases, and combinations thereof, particularly a cellulase having a crystalline:soluble cellulose activity ratio at 60 minutes of at least about 0.1, to clean the porous membrane, and then reusing the porous membrane to continue filtering beer. The present invention further provides a method for producing beer comprising filtering beer through a porous membrane that progressively clogs during filtration, monitoring the streaming or zeta potential of the porous membrane as a measure of the extent of clogging of the porous membrane, halting filtration of the beer through the porous membrane before the porous membrane becomes fully clogged as determined by the streaming or zeta potential of the porous membrane, cleaning the porous membrane, and then reusing the porous membrane to continue filtering beer.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of producing beer,particularly of filtering beer through a filtration medium and cleaningthe filtration medium with enzymes such that it can be reused in beerfiltration.

BACKGROUND OF THE INVENTION

In view of the extended marketing channels, germs (e.g., bacteria) haveto be removed from the beer in order to make it storable. Nowadays, germremoval is mainly carried out by pasteurization of the beer. To thisend, the beer is, for example, bottled or canned, and heated to atemperature of between 62 and 69° C. to kill the germs.

This pasteurization does, however, involve considerable energyconsumption. It has the further disadvantage that the energy introducedcan trigger chemical reactions which impair the product and aredifficult to control. These reactions can, for example, adversely affectthe flavor of the product (“pasteurized taste”), and there is also thedanger that undesired substances will form. Pasteurization is,therefore, a relatively expensive germ removing method involving highenergy expenditure and, consequently, having harmful effects on theenvironment as well as reducing the quality of the product.

Another known germ removing method is cold-filtration. Cold-filteredbeer is available as so-called “draft beer” in, for example, the UnitedStates, Japan and Korea. This beer is prohibited in Europe because itcontains technical enzymes.

These technical enzymes are present in the beer to counteract a drawbackinherent in the cold-filtration method: early clogging of the filter.This clogging is due to deposits of substances to be filtered out of thebeer on the upstream side of the filter, e.g., a membrane filter. Thedeposits are difficult or even impossible to remove from the filter andreduce the service life of the filter. This increases the cost ofproducing the beer as membrane filters are expensive.

To prolong the service life of the filter, the manufacturers of membranefilters recommend cleaning the used membranes by treating them withproteases, glucanases, and xylanases, as well as with chemicals such assurfactants, acids/bases, and oxidizing agents, to make them reusable.This cleaning can be carried out at, for example, two stages, with theabove-mentioned enzymes at a first stage, followed optionally byadditional cleaning with the above-mentioned chemicals in a secondstage.

The literature also discloses methods of cleaning membrane filters usedin filtering beer, which cleaning methods involve a variety oftechniques. For example, U.S. Pat. No. 5,227,819 discloses a method forthe cleaning of a polyamide microporous membrane used in cold-filteringbeer by passing a dilute alkaline solution through the microporousmembrane. International Patent Application WO 96/23579 discloses asomewhat different method of cleaning a membrane filter used in beerfiltration. That method is characterized by treating the membrane filterwith an enzyme-containing aqueous solution of β-glucanases, xylanases,and cellulases, cleaning the membrane filter with an acidic aqueouscleaning solution, and cleaning the membrane filter with aperoxide-containing alkaline cleaning solution.

Given, for example, a filter area of approximately 320 m², a cleaningprocedure will, by way of example, make provision for enzymatic cleaningafter every 5,000 hectoliters filtered and an additional chemicalcleaning after every 20,000 hectoliters filtered. The typical servicelife of filters with the above-mentioned filter area of approximately320 m² having undergone the manufacturer-recommended cleaning isapproximately 100,000 hectoliters.

The previously known cleaning procedures do, however, have thedisadvantage that they are unable to remove the deposits on the filterto a satisfactory extent, which causes the cleaning efficiency todiminish strongly as the membrane filter increases in age.

Yet another disadvantage is the sudden, random clogging of the filtermembrane, unrelated to standard norms like total nitrogen content, orpercent of original wort. A fully clogged membrane filter cannot besatisfactorily cleaned under procedures following the current state oftechnology, which greatly reduces the service life of the filter. It isdifficult to determine when a filter will become so clogged that itcannot be satisfactorily cleaned, and, therefore, a filter may becleaned prematurely or not in time, i.e., too early or too late.

In view of the foregoing problems, there exists a need for an improvedmethod of producing beer, particularly wherein the beer can be filteredthrough a filtration medium that can be satisfactorily cleaned andreused. The present invention provides such a method. These and otheradvantages of the present invention, as well as additional inventivefeatures, will be apparent from the description of the inventionprovided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method for producing beer comprisingfiltering beer through a porous membrane until such time that the porousmembrane is in need of cleaning, contacting the porous membrane with anenzyme selected from the group consisting of cellulases, amylases, andcombinations thereof, particularly a cellulase having acrystalline:soluble cellulose activity ratio at 60 minutes of at leastabout 0.1, to clean the porous membrane, and then reusing the porousmembrane to continue filtering beer. The present invention furtherprovides a method for producing beer comprising filtering beer through aporous membrane that progressively clogs during filtration, monitoringthe streaming or zeta potential of the porous membrane as a measure ofthe extent of clogging of the porous membrane, halting filtration of thebeer through the porous membrane before the porous membrane becomesfully clogged as determined by the streaming or zeta potential of theporous membrane, cleaning the porous membrane, and then reusing theporous membrane to continue filtering beer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of beer filtration amount (g) versus filtration time(sec) in connection with filtering beer through a previously unused,i.e., a new, porous membrane.

FIG. 2 is a graph of beer filtration amount (g) versus filtration time(sec) in connection with filtering beer through a clogged porousmembrane.

FIG. 3 is a graph of beer filtration amount (g) versus filtration time(sec) in connection with filtering beer through a previously cloggedporous membrane cleaned in accordance with a prior art technique.

FIG. 4 is a graph of beer filtration amount (g) versus filtration time(sec) in connection with filtering beer through a previously cloggedporous membrane cleaned in accordance with the present invention.

FIG. 5 is a schematic diagram depicting a device for measuring the zetapotential of a filtration medium.

FIG. 6 is a graph of filtration medium zeta potential (mV) versuselectrolyte solution pH, wherein curve “a” is for a new porous membrane,curve “b” is for a porous membrane that has been partially clogged inconnection with filtering beer, and curve “c” is for a porous membranethat has been nearly fully clogged in connection with filtering beer.

FIG. 7 is a schematic diagram depicting an apparatus for filtering beerusing a bypass system and the measuring device of FIG. 5.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method for producing beer, preferablycold-filtered beer. The method comprises filtering beer through a porousmembrane, i.e., a membrane filter, until such time that the porousmembrane is in need of cleaning, contacting the porous membrane with anenzyme to clean the porous membrane, and then reusing the porousmembrane to continue filtering beer.

It surprisingly has been discovered that porous membranes can be cleanedbetter and more gently with a cellulase and/or with an amylase than withproteases, xylanases, and/or glucanases. Cleaning in accordance with thepresent invention results in a considerable increase in the service lifeof porous membranes used in the filtering of beer and therefore greatlyimproves the commercial benefit attendant the use of porous membranes inthe production of beer.

The enzyme is selected from the group consisting of cellulases,amylases, and combinations thereof. As indicated above, proteases,xylanases, and/or glucanases need not be used, and, preferably, are notused, with the cellulase and/or amylase to clean the porous membrane.The cellulase desirably has a crystalline:soluble cellulose activityratio (described more fully below) of at least about 0.1, more desirablyat least about 0.3, preferably at least about 0.4, more preferably atleast about 0.5, and most preferably at least about 1, particularly atleast about 1.2. Suitable cellulases include cellulases derived fromAspergillus, particularly Aspergillus niger. Preferred cellulasesinclude cellulases derived from Trichoderma, preferably Trichodermareesei and Trichoderma longibrachiatum, and Thermomonospora, preferablyfrom Thermomonospora fusca. Other sources of cellulases are recited inU.S. Pat. No. 4,912,056. Suitable amylases include α-amylase, β-amylase,and combinations thereof. More preferably, no enzymes other thancellulases and amylases are utilized in the present inventive method,i.e., the porous membrane is not contacted with an enzyme other than acellulase or an amylase. Most preferably, the enzyme utilized in thepresent inventive method is a cellulase, and optimally no enzyme otherthan a cellulase is utilized, i.e., the porous membrane is contactedwith a cellulase and is not contacted with any other enzyme.

The porous membrane can be any membrane suitable for the filtration ofbeer. In the context of the present invention, the porous membranetypically will be a microporous membrane, i.e., a porous membrane with apore rating of about 0.02–1 μm. The porous membrane preferably will havea pore rating of about 0.1–1 μm, most preferably about 0.45 μm. Such aporous membrane can be used to remove bacteria and other undesirablegerms from the beer, preferably obviating the need to pasteurize thebeer. The porous membrane also can be used to remove yeast and otherundesirable substances from the beer. Suitable porous membranes includethose prepared from inorganic materials such as ceramics and metals, aswell as, preferably, organic polymers such as polyamides,polyethersulfones, polyolefins, polyvinylidenefluoride, and the like.The porous membrane preferably is a polyamide porous membrane,especially a nylon-6,6 porous membrane.

A preferred embodiment of the method according to the present inventionis characterized in that the porous membrane is additionally broughtinto contact with an aqueous base, with the porous membrane beingadvantageously brought into contact with the aqueous base at a firststage and with the enzyme at a second stage. The use of an aqueoussolution of NaOH and/or KOH as the aqueous base has proven expedient. Itis preferable for the base to be present in a concentration of 0.1 to 1N, more preferably 0.25 to 1 N, and most preferably 0.5 to 1 N. Thetreatment with the aqueous base is best carried out at a temperature ofbetween 40 and 90° C.

Further advantageous embodiments of the method according to the presentinvention are characterized in that the treatment with the cellulase iscarried out at a temperature of between 40 and 50° C. and a pH ofbetween 4.5 and 5.5, the treatment with the α-amylase is carried out ata temperature of between 60 and 75° C. and a pH of between 4.6 and 5.8,and the treatment with the β-amylase is carried out at a temperature ofbetween 40 and 60° C. and a pH of between 4.6 and 5.8.

It is expedient for the cleaning to be carried out until a point in timeat which there is no more change in the streaming potential or the zetapotential of the porous membrane. It has been discovered that thestreaming potential occurring at the porous membrane during operation orthe zeta potential calculated from it (see below) is a good indicationof the extent to which the substances clogging the porous membrane havebeen removed.

The present invention also aims at increasing the porous membrane'sservice life by ensuring that it is cleaned at a desirable time. Thus,the present invention provides for the production of beer comprisingfiltering beer through a porous membrane, which will clog progressivelyas filtration proceeds. Filtration is halted at a given point when theporous membrane is only partially clogged, i.e., has not yet reached thecondition of being totally clogged. The degree of clogging can bedetermined by any suitable means, desirably by monitoring the pressuredrop across the porous membrane such as is generally described in U.S.Pat. No. 5,449,465. Alternatively, the present invention provides for anidentification of the time for cleaning by determination of thestreaming potential through the filter and/or zeta potential of thefilter.

This aspect of the present invention is founded on the recognition thatstreaming potential—or zeta potential extrapolated from the former'srecorded data—will change in a pH range (within which beer brewing orfiltering occurs) according to the degree of clogging and thusrepresents a reliable, and almost quantitative, indicator of the stateof clogging. Determination of the streaming potential and/or zetapotential of the porous membrane can hence give an accurate picture of aparticular state of clogging.

Porous membranes are known to act in a two-fold way. First, a porousmembrane acts as a sieve, when particles larger then the filter's poresare mechanically filtered out of the medium. Secondarily, a porousmembrane also is known to act by electrostatic attraction. Particles ofa diameter much smaller than the pore size of the membrane are depositedthereon when the zeta potential of the filter medium and that of theparticles are of opposite polarity (see, e.g., Informational Brochure SD872h G of Pall Filtrationstechnik GmbH, Germany).

Yet, not known prior to the present invention is the fact that zetapotential can be used to determine a porous membrane's degree ofclogging.

A porous membrane's zeta potential will be affected by its chemicalproperties. One of ordinary skill in the art will have nodifficulty—being cognizant of the present invention—to select onlyfilters whose zeta potential will change at a great enough rate relativeto the degree of clogging. With the filter on-line, and by way ofcontinuous monitoring through data acquisition, the filtration processcan be halted at an appropriate time, e.g., once clogging sets in.

The cleaning of a filter not yet fully clogged is much easier, whileassuring longer service life, than the cleaning of a totally cloggedfilter. Thus, a preferred method of the present invention has filtrationhalted at a point when the filter's zeta potential has decreased to amaximum of 20% of the value it exhibited in its unused state, or whenclogging does not exceed 80%.

Another refinement of the process will use a porous membrane ofpolyamide, with filtration halted when the zeta potential exceeds −5 mVas measured at a pH of 4.2.

The beer preferably will undergo pre-filtration before filtrationproper, i.e., filtration through the porous membrane. Diatomateous (orinfusorial) earth, also known as diatomite, is almost exclusively usedfor pre-filtration. A combination of diatomateous earth and deep-bedfiltration also is feasible.

The present invention can be used in any suitable beer productionsystem. Preferably, the present invention is used in connection with thecluster filter system as described in U.S. Pat. Nos. 5,417,101 and5,594,161.

The present invention also relates to a filtration unit for filteringbeer, with a feeder line for the filtration-bound beer, a porousmembrane, and a run-off line for the filtered beer. It is characterizedby a module in the form of a meter cell, functioning as bypass, andfeaturing a porous membrane and means, e.g., electrodes, for monitoringthe streaming potential and/or zeta potential of the meter cell'smembrane filter through which beer flows.

The present invention also deals with a filtration unit for filteringbeer, with the unit featuring a feeder line for filtration-bound beer, aporous membrane, and a run-off line for filtered beer. In divergencefrom the foregoing paragraph, the filtration unit is characterized bymeans, e.g., electrodes, being attached to the porous membrane formonitoring or reading the streaming potential and/or zeta potential asthe beer flows through the porous membrane. In this variation, the zetapotential is not measured via the meter cell assigned as bypass to themembrane filter, but rather on the membrane filter itself.

Any suitable bypass configuration can be utilized in connection with theembodiments of the present invention. Preferably, the present inventionincorporates the apparatus and method described in U.S. Pat. No.5,449,465.

The discovery that the filter's zeta potential correlates to the generalstate of clogging can be implemented in beer filtration as follows:

1. Through constant observation of changes taking place in the streamingpotential and/or zeta potential of the porous membrane during thefiltration process, the membrane's degree of clogging can be pinpointedin order to prevent an unexpected or random occurrence, while timelymeasures for an exchange of filters can be taken.

2. Filtration can be halted before the porous membrane becomes totallyclogged. This promotes easier cleaning of the filter. It has been shownthat the clogging substances in a totally clogged filter can only beremoved with the greatest of difficulty by conventional methods ofcleansing, or cannot be removed from the filter at all, resulting inabbreviated service life.

Once filtration is halted prior to total clogging, the process ofcleaning is much easier and more thorough, with the filter retaining anextended life. In the instance of a polyamide porous membrane, it hasbeen discovered that the successful removal of all clogging substancesfrom the porous membrane can be accomplished when filtration is haltedat a point where the zeta potential has not lost more than about 80% ofits original value, i.e., is not clogged in excess of 80%.

3. The cleaning method's success can be tested by determining thecleaned membrane's zeta potential. The act of cleaning will return thezeta potential to approximately its original value.

By this procedure, the cleaning process can be evaluated and/oroptimized for it's efficiency:

4. The aging of a porous membrane for reasons of repeated use can betracked, providing a handy estimate as to its remaining service lifeexpectancy.

5. By measuring zeta potential, filter material and shunting materials(e.g., diatomite, bentonite, perlite, polyvinyl pyrrolidone) can betested for suitability in beer filtration by assessing the interactionbetween clogging substances of liquid systems and filter material and/orshunting means for filters.

6. The service life of a porous membrane can be estimated by way ofmeasuring zeta potential, whereunder a specific membrane load (hl/m²) isrecorded up to the point when clogging sets in.

The artisan is aware that most suitable for the process are porousmembranes with a zeta potential exhibiting pronounced change in relationto the degree of clogging. Verification of these parameters is easyenough by employing the aforementioned simple test method.

The following examples further illustrate the present invention but, ofcourse, should not be construed as in any way limiting its scope.

Example 1

This example illustrates the effectiveness of the present inventivemethod to produce beer. In particular, this example demonstrates thatcellulases and amylases can be used to satisfactorily clean a porousmembrane clogged in the course of beer filtration such that the porousmembrane can be reused in continued beer filtration.

A porous membrane made of nylon-6,6 (NB type, commercially availablefrom Pall Filtrationstechnik GmbH, Germany) was used as a filter. Such afilter is frequently used in the state of the art for thecold-filtration of beer.

The so-called membrane filter test according to Esser (Monatszeitschriftfür Brauerei (Monthly Magazine for Breweries) 25^(th) year, No. 6, pages145–151, 1972) was used to determine the filtering performance of thefilter. This test is reliable for checking measures for improvingfilterability.

To determine the filtering efficiency of a new, i.e., unused, porousmembrane, a pressure filtration apparatus (SM 16526 type, 200 mlcapacity; commercially available from Sartorius GmbH, Goettingen,Germany) was used for a polyamide nylon-6,6 porous membrane having a 47mm diameter and a 0.2 μm pore size.

Beer cooled down to 0° C. was forced through the porous membrane underisobaric conditions (1 bar), and the amount of filtrate was weighedevery 10 seconds. The test was stopped after 200 g of filtrate wereobtained. The result is shown as a graph in the diagram of FIG. 1. FIG.1 shows that, under the conditions indicated above, the 200 g offiltrate were obtained with the unused filter after approximately 210seconds.

Under identical conditions, the filtering performance of a partiallyclogged, i.e., used, porous membrane was tested. The result is given inFIG. 2 which shows that even in 720 seconds only approximately 60 g offiltrate were obtained.

The clogged porous membrane was cleaned in accordance with a prior artmethod, wherein the membrane was first cleaned enzymatically and thenchemically, as described below.

For enzymatic cleaning, the clogged membrane was treated for 1 hour witha 1% aqueous solution of a mixture of β-glucanases and xylanases(P3-Ultrasil 65; commercially available from Henkel) with a pH of 5(adjusted with a 0.05% aqueous solution of a mixture of surfactants andan acidic component (P3-Ultrasil 75; commercially available fromHenkel)) at a temperature of 50° C. This treatment was subsequentlycarried out one more time.

The membrane was then treated for 3 hours with a 0.5% aqueous solutionof a mixture of surfactants, glucanases, and proteases (P3-Ultrasil 62;manufacturer: Henkel) with a pH of 9–9.5 (adjusted with a 0.15% aqueoussolution of a mixture of surfactants and an alkaline component(P3-Ultrasil 91; manufacturer: Henkel)) at a temperature of 50° C. andsubsequently rinsed with warm water (50° C.).

For chemical cleaning, the membrane thereafter was treated for 30minutes with a 1% aqueous solution of a mixture of surfactants and anacidic component (P3-Ultrasil 75; commercially available from Henkel) at60° C., and then rinsed with fresh water. The membrane was subsequentlytreated for 30 minutes with an aqueous solution containing 1% of amixture of surfactants and an alkaline component (P3-Ultrasil 91;commercially available from Henkel) and 1% of a mixture of surfactantsand an oxygen donor (P3-Ultrasil 05; commercially available from Henkel)at a temperature of 60° C. and then rinsed with fresh water. Themembrane was then treated once more for 30 minutes with a 0.5% aqueoussolution of a mixture of surfactants and an acidic component(P3-Ultrasil 75; commercially available from Henkel) and subsequentlyrinsed with fresh water until the rinse water reached the electricalconductivity of fresh water.

The filtering performance of this cleaned porous membrane was thentested again under the conditions indicated above. The result is shownin FIG. 3. FIG. 3 shows that the filtering performance has improvedsomewhat as the 200 g of filtrate were obtained after approximately 600seconds.

A similarly clogged membrane whose filtering efficiency is shown in FIG.2 was cleaned in accordance with the method according to the presentinvention. The membrane was treated for 30 minutes with an aqueoussolution of C₁- and C_(x)-cellulases, the solution having a pH value of4.7, at a temperature of 45° C. The membrane was then treated with thesame solution, but at a pH value of 5.0 and a temperature of 50° C.,and, finally, at a pH value of 4.7 and a temperature of 60° C. for 60minutes.

The membrane was subsequently rinsed with warm water at 50° C. Thefiltering performance of the membrane cleaned in accordance with thepresent invention was tested in accordance with the above procedure. Theresult is shown in FIG. 4.

FIG. 4 shows that 200 g of filtrate were obtained after approximately220 seconds. This represents a significant improvement over the priorart (FIG. 3). The method according to the present invention, therefore,allows considerably better cleaning of a used membrane filter than ispossible with prior art cleaning methods.

Equally good results were obtained when, in accordance with the presentinvention, an amylase was used instead of a cellulose. The service lifeof a porous membrane thus can be increased with the cleaning methodaccording to the present invention.

Example 2

This example illustrates the use of the streaming or zeta potential of aporous membrane to assist in the cleaning of the porous membrane. Inparticular, the streaming or zeta potential is demonstrated to be usefulin determining the extent of membrane cleaning as well as when amembrane is most satisfactorily cleaned.

The zeta potential of membrane filters was determined with theelectrokinetic measuring system EKA of Anton Paar GmbH, Austria. Thismeasurement is based on the streaming potential method. An electrolyteflows through the filters, and the potential (streaming potential) whichis produced by shearing-off of counterions is detected with electrodes,and the zeta potential is calculated from this measured quantity (seebelow).

FIG. 5 shows diagrammatically the measuring cell with which thestreaming potential or the zeta potential was determined. Referencenumeral 1 designates the measuring cell in which the porous membrane 2is clamped without warping in filter holders 3 and 4 made ofpolytetrafluoroethylene. The filter holders 3 and 4 are the end piecesof two pistons 5 and 6, respectively, which are mounted for displacementin the cylindrical part 7 of the measuring cell 1.

The end pieces 3 and 4 of the pistons 5 and 6, respectively, have finebores 10 and 11 for the fluid which is to be filtered and press theperforated electrodes 8 and 9 against the porous membrane 2. Theelectrodes 8 and 9 are connected to the two electric terminals 12 and 13extending inside the pistons 5 and 6 so the streaming potential built upas fluid flows through the membrane 2 can be measured. Silver electrodesor silver chloride electrodes which exhibit a low polarization duringpassage of current are preferred for the electrodes. The pistons 6 and 7are mounted in the seals 14 and 15, respectively, such that, on the onehand, they are displaceable, and, on the other hand, they do not allowany fluid to leak from the measuring cell.

The fluid to be filtered flows through the supply line 16 into thecylindrical part 7 of the measuring cell 1, through the fine bores 10 ofthe piston 6, through the electrode 8, with an electric potential beingbuilt up, and through the porous membrane 2. The filtered fluid flowsthrough the electrode 9, with a potential likewise being built up,passes through the fine bores 11 of the piston and leaves the measuringcell through the discharge line 17.

To determine the zeta potential from the measured streaming potential,measurement (not illustrated) of the differential pressure in themeasuring cell between supply line 16 and discharge line 17, theconductivity and also the pH value is necessary. The zeta potential iscalculated from these measured quantities as follows:

${{zeta}\mspace{14mu}{potenital}} = {\frac{U}{\Delta\; p} \cdot \frac{{LF} \cdot \eta}{ɛ \cdot {ɛ{^\circ}}}}$where U is the streaming potential, Δp the pressure difference, LF theconductivity, η the viscosity, and ∈∈ the dielectric constant.

The change in the zeta potential of the membrane filter as cloggingprogresses is shown in FIG. 6. This figure is a diagram in which thezeta potential in millivolts is plotted as ordinate, and the pH value atwhich the zeta potential was determined as abscissa. The pH value of theelectrolyte solution (0.001 N aqueous KCl solution) was set with 0.1 NHCl or with 0.1 N NaOH. The specified pressure difference was 350 mbar.

The diagram was obtained by first determining with the measuring celldescribed above the zeta potentials of a new, i.e., unused porousmembrane made of polyamide (NB type, commercially available from PallFiltrationstechnik GmbH, 6072 Dreieich 1, Germany) at various pH values.

The results relating to the unused porous membrane are plotted as curve“a”. It is evident that the unused porous membrane has a zeta potentialof approximately −18 mV with an alkaline pH, and that the zeta potentialincreases with decreasing pH and finally reaches zero value at a pH ofapproximately 3.

Curve “b” shows the dependence of the zeta potential on the pH value ofthe porous membrane under identical measuring conditions, as statedabove, but after use thereof for filtering beer and, therefore, withpartial clogging. As is apparent, the zeta potential is raised somewhatby the partial clogging and only reaches a value of approximately −15 mVat pH values of approximately 7.

Curve “c” was plotted for the same porous membrane in the nearly fullyclogged state. It is evident that the zeta potential now changes onlyslightly with the pH value, and even in the alkaline range does not fallbelow approximately −2 mV.

To test the cleaning according to the present invention, the zetapotential of the membrane to be cleaned is determined, and the cleaningwas successful if the zeta potential of the cleaned membrane shifted asfar as possible in the direction of the zeta potential of the unusedmembrane.

It will be clear to one skilled in the art that porous membranes whosezeta potential changes to a sufficiently great extent as a function ofthe degree of clogging are particularly well-suited for use in themethod according to the present invention. This characteristic can beeasily determined by one skilled in the art by simple testing.

A porous membrane of polyamide is especially suitable in the context ofthe process since the zeta potential at the pH of the filtration-boundbeer (ca. pH=4.2) will undergo severe change with progressive clogging.As can be learned from FIG. 6, the membrane at this particular pH valueat the beginning of filtration shows a zeta potential of approximately−8 mV. The totally clogged membrane has a zeta potential ofapproximately −2 mV.

FIG. 7 shows a variation of the discussed filtration unit featuring afiltration chamber 18, with a meter cell 22 assigned to it as a bypass,as depicted in FIG. 5. The filtration chamber 18 holds filter candles19.

The filtration-bound beer is fed via line 20 into the filtration chamber18, flowing through the filter candles (membrane filter) 19, and exitsthe filter chamber 18 through run-off line 21 in the form of filteredbeer.

The meter cell is shown in FIG. 7 without detail. The actual flowthrough the meter cell 22 must be controlled to the extent that anamount of beer is filtered per cm² of the porous membrane's surfacewhich is equal to the amount of porous membrane surface per cm² in thefiltration chamber 18.

The severe change in zeta potential of the filter membrane 2 (FIG. 5)inside meter cell 1 during filtration allows an assessment of the stateof the filter candles 19 in filtration chamber 18.

Example 3

This example illustrates the effectiveness of cellulase derived fromAspergillus niger in enzymatically degrading soluble and crystallinecellulose substrates.

Cellulase derived from Aspergillus niger was obtained from Fluka (itemnumbers 22178). The enzyme was evaluated with respect to two differentcelluloses: soluble carboxymethylcellulose (CMC, available from Aldrichas item number 41927-3) and crystalline cellulose (Avicel, availablefrom FMC as item number PH-105).

The test methodology involved the preparation of an incubation solutionof (i) 18 ml CMC (1%) or Avicel (1%), (ii) 5 ml sodium acetate buffer(50 mM, pH 4.8), and (iii) 5 ml of a solution of the enzyme in sodiumacetate buffer (50 mM, pH 4.8) at 30° C. A test solution then wasprepared by mixing 1.4 ml of the incubation solution with 0.1 ml glucosesolution (0.15%) and 1.5 ml 3,5-dinitrosalicylic acid (DNS) reagent(available from Sigma as item number D-0550). The test solution wasboiled for 15 minutes. The total μmol glucose equivalents/mg enzyme as afunction of time (min) was determined spectroscopically (575 nm), usingtwo parallel samples, in accordance with the procedure described inMiller, Anal. Chem., 31, 426–28 (1959), using a straight calibrationwith a glucose standard. Protein amounts were determined in accordancewith the procedure described in Bradford, Anal. Biochem., 72, 248–64(1976), using a bovine serum albumin (BSA) standard.

The enzymatic degradation of cellulose results in the production ofglucose, and, therefore, the measurement of μmol glucose equivalents/mgenzyme is a measure of the activity of the enzyme with respect to aparticular type of cellulose, e.g., soluble (CMC) or crystalline(Avicel) cellulose.

The results of this evaluation with respect to the cellulase derivedfrom Aspergillus niger are set forth in Table 1. The test solution withthe soluble (CMC) cellulose substrate contained 0.8 mg enzyme/28 mlincubation solution (ca. 17.6 μg protein). The test solution with thecrystalline (Avicel) cellulose substrate contained 0.35 mg enzyme/28 mlincubation solution (ca. 7.7 μg protein).

TABLE 1 Cellulase derived from Aspergillus niger Glucose Equivalents(μmol/mg enzyme) Crystalline: Soluble Crystalline Soluble CelluloseCellulose Cellulose Time (min) Substrate Substrate Activity Ratio 0 00   — 10 27.0 0   0   15 28.5 1.7 0.06 30 30.5 — — 45 34.0 1.7 0.05 6034.8 4.0 0.11 75 37.5 3.5 0.09 90 37.8 3.7 0.10 105 38.3 — — 120 39.510.3  0.26

Those enzymes that have a relatively greater activity toward crystallinecellulose substrates as compared to soluble cellulose substrates havebeen found to be particularly effective in cleaning porous membranesused in beer filtration. The ratio of the glucose equivalents producedwith respect to the crystalline cellulose substrate and the glucoseequivalents produced with respect to the soluble cellulose substratethus is an indicator of the effectiveness of the enzyme in the contextof the present invention and is described as the crystalline:solublecellulose activity ratio. Desirably, the crystalline:soluble celluloseactivity ratio has the previously described values at a range of timesin the test protocol described in this example, e.g., at 30 minutes, 60minutes, and/or 90 minutes, especially at 60 minutes.

As is apparent from the data set forth in Table 1, the cellulase fromAspergillus niger has a crystalline:soluble cellulose activity ratio at60 minutes of 0.11, indicating that it is a moderately effective enzymefor purposes of cleaning porous membranes used in connection with thefiltration of beer.

Example 4

This example illustrates the effectiveness of cellulase derived fromTrichoderma reesei in enzymatically degrading soluble and crystallinecellulose substrates.

Cellulase derived from Trichoderma reesei was obtained from Fluka (itemnumbers 22173). The enzyme was evaluated in the same manner as recitedin Example 3.

The results of this evaluation with respect to the cellulase derivedfrom Trichoderma reesei are set forth in Table 2. The test solution withthe soluble (CMC) cellulose substrate contained 0.37 mg enzyme/28 mlincubation solution (ca. 128 μg protein). The test solution with thecrystalline (Avicel) cellulose substrate contained 0.08 mg enzyme/28 mlincubation solution (ca. 25.6 μg protein).

TABLE 2 Cellulase derived from Trichoderma reesei Glucose Equivalents(μmol/mg enzyme) Crystalline: Soluble Crystalline Soluble CelluloseCellulose Cellulose Time (min) Substrate Substrate Activity Ratio 0 0 0— 5 62.6 0 — 10 84.7 21.5 0.25 15 96.3 30.0 0.31 30 99.5 40.0 0.40 45152.0 57.5 0.38 60 139.0 75.0 0.54 75 178.4 85.0 0.48 90 184.2 95.0 0.52105 172.6 100.0 0.58 120 193.7 115.0 0.59

As is apparent from the data set forth in Table 2, the cellulase fromTrichoderma reesei has a crystalline:soluble cellulose activity ratio at60 minutes of 0.54, indicating that it is a superior enzyme for purposesof cleaning porous membranes used in connection with the filtration ofbeer.

Example 5

This example illustrates the effectiveness of cellulase derived fromBacillus subtilis in enzymatically degrading soluble and crystallinecellulose substrates.

β-cellulase derived from Bacillus subtilis was obtained from Fluka (itemnumbers 49106). The enzyme was evaluated in the same manner as recitedin Example 3.

The results of this evaluation with respect to the β-cellulase derivedfrom Bacillus subtilis are set forth in Table 3. The test solution withthe soluble (CMC) cellulose substrate contained 14.4 mg enzyme/28 mlincubation solution (ca. 8.3 μg protein). The test solution with thecrystalline (Avicel) cellulose substrate contained 15.6 mg enzyme/28 mlincubation solution (ca. 8.8 μg protein).

TABLE 3 β-Cellulase derived from Bacillus subtilis Glucose Equivalents(μmol/mg enzyme) Crystalline: Soluble Crystalline Soluble CelluloseCellulose Cellulose Time (min) Substrate Substrate Activity Ratio 0 0 0— 5 1.1 0.1 0.09 10 1.0 0.1 0.10 15 0.9 0.1 0.11 30 0.9 0.1 0.11 45 1.00.1 0.10 60 1.0 0.1 0.10 75 1.0 0.2 0.20 90 1.1 0.2 0.18 105 1.1 0.10.09 120 1.3 0.1 0.08

As is apparent from the data set forth in Table 3, the β-cellulase fromBacillus subtilis has a crystalline:soluble cellulose activity ratio at60 minutes of 0.10, indicating that it is a moderately effective enzymefor purposes of cleaning porous membranes used in connection with thefiltration of beer.

Example 6

This example illustrates the effectiveness of exocellulase derived fromThermomonospora fusca in enzymatically degrading soluble and crystallinecellulose substrates.

Exocellulase E3 derived from Thermomonospora fusca was obtained fromCornell University. The enzyme was evaluated in the same manner asrecited in Example 3 except that the incubation solution comprised (i)18 ml CMC (1%) or Avicel (1%), (ii) 9 ml sodium acetate buffer (50 mM,pH 5.6), and (iii) 1 ml of a solution of the enzyme in sodium acetatebuffer (50 mM, pH 5.6), shaken at 50° C. (ca. 960 μm protein). The testsolution was evaluated using a color test rather than the DNS testrecited in Example 3.

The results of this evaluation with respect to the exocellulase derivedfrom Thermomonospora fusca are set forth in Table 4.

TABLE 4 Exocellulase derived from Thermomonospora fusca GlucoseEquivalents (μmol/mg enzyme) Crystalline: Soluble Crystalline SolubleCellulose Cellulose Cellulose Time (min) Substrate Substrate ActivityRatio 0 0 0 — 5 0.1 0 — 10 0.1 0.3 3.00 15 0.2 0.3 1.50 30 0.2 0.3 1.5045 0.3 0.4 1.33 60 0.3 0.4 1.33 75 0.3 0.5 1.67 90 0.3 0.3 1.00

As is apparent from the data set forth in Table 4, the Exocellulasederived from Thermomonospora fusca has a crystalline:soluble celluloseactivity ratio at 60 minutes of 1.33, indicating that it is a superiorenzyme for purposes of cleaning porous membranes used in connection withthe filtration of beer.

Example 7

This example illustrates the effectiveness of α-amylase derived fromBacillus subtilis in enzymatically degrading soluble and crystallinecellulose substrates.

α-amylase derived from Bacillus subtilis was obtained from Fluka (itemnumbers 10069). The enzyme was evaluated in the same manner as recitedin Example 3 except that the incubation solution comprised (i) 18 ml CMC(1%) or Avicel (1%), (ii) 5 ml sodium acetate buffer (50 mM, pH 6.9),and (iii) 5 ml of a solution of the enzyme in sodium acetate buffer (50mM, pH 6.9), shaken at 30° C. (ca. 8.5 μm protein). The test solutionwas evaluated using a color test rather than the DNS test recited inExample 3.

The results of this evaluation with respect to the α-amylase derivedfrom Bacillus subtilis are set forth in Table 5.

TABLE 5 α-Amylase derived from Bacillus subtilis Glucose Equivalents(μmol/mg enzyme) Crystalline: Soluble Crystalline Soluble CelluloseCellulose Cellulose Time (min) Substrate Substrate Activity Ratio 0 0 0— 5 0.1 0 0 10 0.1 0 0 15 0.1 0 0 30 0.1 0 0 45 0.1 0 0 60 0.1 0 0 750.1 0 0 90 — 0 —

As is apparent from the data set forth in Table 5, the α-amylase fromBacillus subtilis has a crystalline:soluble cellulose activity ratio at60 minutes of about 0 (<0.1 μmol detection limit), indicating that it isnot as effective an enzyme for purposes of cleaning porous membranesused in connection with the filtration of beer as the previouslydescribed cellulases.

Example 8

This example illustrates the effectiveness of various cellulases inenzymatically degrading soluble and crystalline cellulose substrates.

Cellulase preparations were obtained from the Erbsloh Company: (a)C_(x)-cellulase (powder, item number VP 0945/2), (b) C₁-cellulase fromTrichoderma reesei (powder, item number VP 0965/2), (c) C₁-cellulase(liquid, item number Cleanzym SB1), (d) C₁-cellulase (liquid, itemnumber VP 0976/4), (e) cellulase (liquid, item number VP 0971/1), and(f) cellulase (liquid, item number VP 0971/4). The enzymes wereevaluated in a manner similar to that recited in Example 3 except thatthe incubation solutions comprised (i) 23 ml CMC (1%) or Avicel (1%) ina sodium acetate buffer (50 mM, pH 4.8), and (ii) 5 ml of a solution ofthe enzyme in sodium acetate buffer (50 mM, pH 4.8). 0.5% stocksolutions were prepared from the powdered enzyme preparations (5 mg/ml)and liquid enzyme preparations (5 μl/ml). The solutions were shaken at30° C. The test solution was evaluated after making a 1:5 dilution usinga color test rather than the DNS test recited in Example 3.

The results of this evaluation with respect to the various cellulasesare set forth in Table 6. The glucose equivalents data is in terms ofaverage μmol glucose equivalents/min (for the total time interval) andare not normalized per mg enzyme (as was the situation with the datarecited in Tables 1–5). The crystalline:soluble cellulose activityratio, of course, is not altered by the units for the glucoseequivalents inasmuch as the units divide out in calculating the ratio(i.e., the ratio is unit-less).

TABLE 6 Cellulase Preparations Glucose Equivalents (μmol/min)Preparation (a) Preparation (b) Preparation (c) Crystalline:Crystalline: Crystalline: Soluble Soluble Soluble Soluble CrystallineCellulose Soluble Crystalline Cellulose Soluble Crystalline CelluloseCellulose Cellulose Activity Cellulose Cellulose Activity CelluloseCellulose Activity Time (min) Substrate Substrate Ratio SubstrateSubstrate Ratio Substrate Substrate Ratio 0 0 0 — 0 0 — 0 0 — 5 0.12 0 00.14 0.11 0.79 0.09 0.05 0.56 10 0.06 0.01 0.17 0.11 0.08 0.73 0.09 0.060.67 15 0.06 0.01 0.17 0.08 0.09 1.13 0.08 0.04 0.50 30 0.02 0.02 1.000.07 0.05 0.71 0.07 0.03 0.43 45 0.04 0.02 0.50 0.06 0.04 0.67 0.03 0.020.67 60 0.03 0.02 0.67 0.04 0.03 0.75 0.05 0.02 0.40 75 0.03 0.02 0.670.04 0.03 0.75 0.04 — 90 0.02 0.01 0.50 0.03 0.03 1.00 0.03 0.2 0.67Glucose Equivalents (μmol/min) Preparation (d) Preparation (e)Preparation (f) Crystalline: Crystalline: Crystalline: Soluble SolubleSoluble Soluble Crystalline Cellulose Soluble Crystalline CelluloseSoluble Crystalline Cellulose Cellulose Cellulose Activity CelluloseCellulose Activity Cellulose Cellulose Activity Time (min) SubstrateSubstrate Ratio Substrate Substrate Ratio Substrate Substrate Ratio 0 00 — 0 0 — 0 0 — 5 0.01 0.23 23.0 0.15 0.13 0.87 0.07 0.04 0.57 10 0.190.14 0.74 0.10 0.09 0.90 0.06 0.03 0.50 15 0.15 0.12 0.80 0.06 0.05 0.830.04 0.02 0.50 30 0.10 0.09 0.90 0.05 0.03 0.60 0.03 0.01 0.33 45 0.070.07 1.00 0.04 0.03 0.75 0.02 0.01 0.50 60 0.06 0.06 1.00 0.03 0.02 0.670.02 0.01 0.50 75 0.05 0.06 1.20 0.03 0.02 0.67 0.02 0.01 0.50 90 0.040.06 1.50 0.03 0.02 0.67 0.01 0.01 1.00

As is apparent from the data set forth in Table 6, the variouscellulases have crystalline:soluble cellulose activity ratios at 60minutes ranging from 0.4–1.0, indicating that they are superior enzymesfor the purpose of cleaning porous membranes used in connection with thefiltration of beer.

Example 9

This example further illustrates the effectiveness of the presentinventive method to produce beer. In particular, this exampledemonstrates that cellulases alone (i.e., without the use of otherenzymes) are superior in the cleaning of porous membranes clogged in thecourse of beer filtration for the purpose of returning the porousmembrane to use in continued beer filtration.

Beer of different characteristics was filtered through nylon-6,6 porousmembranes (ca. 300 m²) with a pore rating of 0.45 μm in a cluster filterarrangement (PALL-CFS, available from Pall Filtrationstechnik GmbH,Germany). At certain beer filtration intervals, the porous membraneswere subjected to a cleaning process in accordance with the presentinvention.

The cleaning process involved circulation of a 0.5% NaOH solution for 15minutes, followed by a 60 minute soak. The porous membranes then werebackflushed with water. An internal loop was established through thecluster filter arrangement with water at 38° C. Lactic acid was added tothe water to adjust the pH to 4.2±0.3, and then 6 l of an enzymepreparation containing a cellulase derived from Trichodermalongibrachiatum obtained from the Erbsloh Company (item number VP0945/1) was added to the water via a dosing pump. The enzyme preparationin the water (at a concentration of about 20–40 g enzyme/100 kg filterhousing fluid volume) was circulated for about 15 minutes, followed by a30 minute soak, another 15 minute circulation, and finally a 6 hoursoak. The porous membranes then were backflushed with water.

The porous membranes were cleaned after about 90,000 hl total beer wasfiltered through the porous membranes, and then the porous membraneswere returned to service, i.e., to continue filtering beer. The porousmembranes similarly were cleaned and returned to service after about100,000 hl, about 140,000 hl, and about 165,000 hl total beer wasfiltered through the porous membranes. The porous membranes mechanicallyfailed after about 190,000 hl total beer was filtered through the porousmembranes.

The foregoing data demonstrates that beer can be satisfactorily producedusing the present invention. Specifically, the results of this exampledemonstrate that a porous membrane can be effectively cleaned andreturned to service in accordance with the present invention, therebyprolonging the useful life of the porous medium in a beer productionprocess.

All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments may be used and that it isintended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the following claims.

1. A method for producing beer comprising: (a) filtering beer through aporous membrane until such time that the porous membrane is in need ofcleaning, (b) contacting the porous membrane with an enzyme selectedfrom the group consisting of cellulases, amylases, and combinationsthereof in the absence of any other enzymes to clean the porousmembrane, and (c) then reusing the porous membrane to continue filteringbeer.
 2. The method of claim 1, wherein the porous membrane is contactedwith the cellulase and no other enzyme.
 3. A method for producing beercomprising: (a) filtering beer through a porous membrane until such timethat said porous membrane is in need of cleaning, (b) contacting theporous membrane with a cellulase, in absence of any other enzymes, thecellulase having a crystalline:soluble cellulase activity ratio at 60minutes of at least about 0.1 to clean the porous membrane, and (c) thenreusing the porous membrane to continue filtering beer.
 4. The method ofclaim 3, wherein the cellulase has a crystalline:soluble celluloseactivity ratio at 60 minutes of at least about 0.3.
 5. The method ofclaim 4, wherein the cellulase has a crystalline:soluble celluloseactivity ratio at 60 minutes of at least about 0.4.
 6. The method ofclaim 5, wherein the cellulase has a crystalline:soluble celluloseactivity ratio at 60 minutes of at least about 0.5.
 7. The method ofclaim 6, wherein the cellulase has a crystalline:soluble celluloseactivity ratio at 60 minutes of at least about
 1. 8. The method of claim7, wherein the cellulase has a crystalline:soluble cellulose activityratio at 60 minutes of at least about 1.2.
 9. The method of claim 3,wherein the cellulase is derived from Trichoderma.
 10. The method ofclaim 9, wherein the Trichoderma is Trichoderma reesei or Trichodermalongibrachiatum.
 11. The method of claim 3, wherein the cellulase isderived from Thermomonospora.
 12. The method of claim 11, wherein theThermomonospora is Thermomonospora fusca.
 13. The method of claim 1,wherein the porous membrane is contacted with an amylase.
 14. The methodof claim 13, wherein the amylase is selected from the group consistingof α-amylase, β-amylase, and the combination thereof.
 15. The method ofclaim 3, wherein the method further comprises contacting the porousmembrane with an aqueous base prior to reusing the porous membrane. 16.The method of claim 15, wherein the aqueous base is an aqueous solutionof NaOH and/or KOH.
 17. The method of claim 15, wherein the base ispresent in a concentration of 0.1–1 N in the aqueous base.
 18. Themethod of claim 15, wherein the porous membrane is contacted with theaqueous base at a temperature of 40–90° C.
 19. The method of claim 1,wherein the porous membrane is contacted with α-amylase at a temperatureof 60–75° C. and a pH of 4.6–5.8.
 20. The method of claim 1, wherein theporous membrane is contacted with β-amylase at a temperature of 40–60°C. and a pH of 4.6–5.8.
 21. The method of claim 3, wherein the porousmembrane is cleaned until the zeta potential of the porous membraneceases to change.
 22. The method of claim 3, wherein the time that theporous membrane is in need of cleaning is determined by the pressuredrop across the porous membrane.
 23. The method of claim 3, wherein themethod further comprises determining the time that the porous membraneis in need of cleaning by determining the streaming potential or zetapotential of the porous membrane.
 24. The method of claim 23, whereinthe filtration is halted when the streaming potential or zeta potentialof the porous membrane is reduced to 20% of its original value for theunused porous membrane.
 25. The method of claim 3, wherein the porousmembrane is a polyamide porous membrane.
 26. The method of claim 25,wherein the filtration is halted when the zeta potential of the porousmembrane exceeds −5 mV as measured at pH 4.2.
 27. The method of claim 3,wherein the filtering of the beer is cold-filtering of the beer.
 28. Themethod of claim 1, wherein contacting the porous membrane with an enzymecomprises contacting the porous membrane with a cellulase having acrystalline:soluble cellulase activity ratio at 60 minutes of at leastabout 0.1 to clean the porous membrane.
 29. The method of claim 3,wherein the porous membrane is a nylon-6,6 membrane.
 30. The method ofclaim 3, wherein the porous membrane has a pore rating of about 0.02–1μm.
 31. The method of claim 30, wherein the porous membrane has a porerating of about 0.1–1 μm.
 32. The method of claim 31 wherein the porousmembrane has a pore rating of about 0.45 μm.
 33. The method of claim 3,wherein the method further comprises pre-filtering the beer beforefiltering the beer through the porous membrane.
 34. The method of claim33, wherein the beer is pre-filtered through Diatomateous earth or acombination of Diatomateous earth and deep-bed filtration.